Anal. Chem. 1997, 69, 1525-1529
Hydrogen/Deuterium Exchange To Differentiate Fragment Ions from Pseudomolecular Ions by Electrospray Tandem Mass Spectrometry Adeboye Adejare* and Paul W. Brown
Division of Pharmaceutical Sciences, School of Pharmacy, University of MissourisKansas City, Kansas City, Missouri 64110
Fragmentation of protonated molecules can occur for some compounds even when using gentle electrospray ionization conditions. For many compounds, the fragment ions generated during desolvation (in-source dissociation) cannot be distinguished from protonated molecules (pseudomolecular ions), such as an impurity, decomposition product, metabolite, or reaction byproduct, which have the same structure. This application of hydrogen/deuterium (H/D) exchange allows fragment ions formed from in-source dissociation to be distinguished from pseudomolecular ions using H/D exchange. This technique is based on fragmentation processes which involve neutral losses arising from cleavage of carbonheteroatom bonds. In these situations, the fragment ion does not have all active hydrogens exchanged and can be differentiated from a fully deuterium-exchanged molecule by 1 Da. For some analyses, the method being reported may be more suitable than chromatographic methods.
Determination of the number and position of exchangeable (active) hydrogens serves as a useful method for elucidating molecular structure. Instrumental techniques such as nuclear magnetic resonance (NMR) and mass spectrometry (MS) are critical in such determinations. In the area of MS, hydrogen/ deuterium (H/D) exchange experiments have facilitated structural elucidation and interpretation of fragmentation processes. For nearly every ionization/sample introduction method, a procedure has been developed to perform H/D exchange experiments. The various ionization/sample introduction methods include electron impact (EI),1-3 chemical ionization (CI),4-10 atmospheric pressure (1) McCloskey, J. A. In Methods in Enzymology; McCloskey, J. A., Ed.; Academic: New York, 1990; Vol. 193, pp 329-338. (2) Budzikiewicz, H.; Djerassi, C.; Williams, D. H. Structure Elucidation of Natural Products by Mass Spectrometry; Holden-Day: San Francisco, CA, 1964; Vol. 1, pp 17-40. (3) Biemann, K. Mass Spectrometry Organic Chemical Applications; McGrawHill: New York, 1962; pp 204-250. (4) Hunt, D. F.; McEwen, C. N.; Upham, R. A. Tetrahedron Lett. 1971, 47, 4539-4542. (5) Harrison, A. G. Chemical Ionization Mass Spectrometry; CRC Press: Boca Raton, FL, 1983; pp 129-149. (6) Martinsen, D. P.; Buttrill, S. E., Jr. Org. Mass Spectrom. 1976, 11, 762772. (7) Mueller, D. R.; Eckersley, M.; Richter, W. J. Org. Mass Spectrom. 1988, 23, 217-222. (8) Khaselev, N.; Mandelbaum, A. J. Mass Spectrom. 1995, 30, 1533-1538. (9) Hunt, D. F.; McEwen, C. N.; Upham, R. A. Anal. Chem. 1972, 44, 12921294. (10) Henion, J. D. J. Chromatogr. Sci. 1981, 19, 57-64. S0003-2700(96)01030-X CCC: $14.00
© 1997 American Chemical Society
chemical ionization (APCI),11 fast atom bombardment (FAB),12-16 thermospray,17,18 gas chromatography/mass spectrometry (GC/ MS),19 and electrospray ionization (ESI).20-26 Conformational changes, for example in proteins, have also been probed by ESI using both solution- and gas-phase H/D exchange methods.22-25,27,28 We now report a new use of H/D exchange, using electrospray tandem MS, to differentiate fragment and pseudomolecular ions which have the same structure. ESI is one of the softest means of producing ions for introduction into a mass spectrometer. Desolvation (declustering) of the analyte ions is facilitated by passing through a heated capillary and/or by collisional activation.29 Collisional activation is achieved by an electrostatic field in the intermediate pressure region between the capillary exit and skimmer cone. The ion current generated is a function of capillary temperature, declustering potential, and intrinsic response for that compound. During desolvation of the analyte ions, in-source collisionally induced dissociation (CID) of pseudomolecular ions can occur. Pseudomolecular ions and ions generated by CID are usually even-electron ions. In general, even-electron ions will fragment (11) Kenny, D. V.; Olesik, S. V. J. Am. Soc. Mass Spectrom. 1994, 5, 544-552. (12) Cushnir, J. R.; Naylor, S.; Lamb, J. H.; Farmer, P. B. Rapid Commun. Mass Spectrom. 1990, 4, 426-431. (13) Kenny, P. T. M.; Nomoto, K; Orlando, R. Rapid Commun. Mass Spectrom. 1992, 6, 95-97. (14) Sethi, S. K.; Smith, D. L.; McCloskey, J. A. Biochem. Biophys. Res. Commun. 1983, 112, 126-131. (15) Verma, S.; Pomerantz, S. C.; Sethi, S. K.; McCloskey, J. A. Anal. Chem. 1986, 58, 2898-2902. (16) The´venon-Emeric, G.; Kozlowski, J.; Zhang, Z.; Smith, D. L. Anal. Chem. 1992, 64, 2456-2458. (17) Edmonds, C. G.; Pomerantz, S. C.; Hsu, F. F.; McCloskey, J. A. Anal. Chem. 1988, 60, 2314-2317. (18) Siegel, M. M. Anal. Chem. 1988, 60, 2090-2095. (19) Mahmoud, M. E.; Moussa, A. M.; Forsyth, D. A.; Vouros, P. J. Chromatogr. 1991, 549, 416-422. (20) Hemling, M. E.; Conboy, J. J.; Bean, M. F.; Mentzer, M.; Carr, S. A. J. Am. Soc. Mass. Spectrom. 1994, 5, 434-442. (21) Sepetov, N. F.; Issakova, O. L.; Lebl, M.; Swiderek, K.; Stahl, D. C.; Lee, T. D. Rapid Commun. Mass Spectrom. 1993, 7, 58-62. (22) Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R. T.; Dobson, C. M. Science 1993, 262, 895-900. (23) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1993, 115, 6317-6321. (24) Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1991, 5, 214-217. (25) Anderegg, R. J.; Wagner, D. S.; Stevenson, C. L.; Borchardt, R. T. J. Am. Soc. Mass. Spectrom. 1994, 5, 425-433. (26) Hopfgartner, G.; Vetter, W.; Meister, W.; Ramuz, H. J. Mass Spectrom. 1996, 31, 69-76. (27) Miranker, A.; Robinson, C. V.; Radford, S. E.; Dobson, C. M. FASEB J. 1996, 10, 93-101. (28) Wood, T. D.; Chorush, R. A.; Wampler F. M.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2451-2454. (29) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451.
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into lower molecular mass even-electron ions.30 Decomposition of an even-electron ion to an odd-electron ion is usually energetically unfavorable. Thus, fragment ions generated in an electrospray source usually arise from loss of an even-electron neutral species from a protonated molecule. For many compounds, fragment ions generated during desolvation (in-source dissociation) cannot be distinguished from protonated molecules (pseudomolecular ions), such as an impurity, decomposition product, or reaction byproduct, which have the same chemical structure. This application of H/D exchange allows in-source dissociation to be distinguished from pseudomolecular ions, and consequently the molecular ions, using H/D exchange. This technique is based on fragmentation processes which involve neutral losses arising from cleavage of carbon-heteroatom bonds. The neutral loss eliminated from a parent ion can create a fragment ion indistinguishable from a protonated molecule with the same structure. To our best knowledge, this is the first report of the use of H/D exchange to differentiate fragment ions from such pseudomolecular ions. We illustrate this new technique with an example where the active hydrogens are first exchanged with deuterium. The leaving group transfers a (nonactive) hydrogen to the fragment ion, and the neutral loss is the equivalent of an alkene.
When the active hydrogens are exchanged for deuteriums in the parent ion, the fragment ion produced by CID has a different number of deuteriums when compared to a deuteron adduct completely exchanged molecule (MD) generated from solution. The fragment ion in this situation has one less deuterium than a fully exchanged MD desolvated ion from solution. Thus, origins of two ions which have the same molecular structure, whether generated from the gas phase or solution, can be differentiated on the basis of mass. This new method is useful when elucidating structures of organic compounds, especially amines and alcohols. It can also be used in determining purity of such compounds. EXPERIMENTAL SECTION The compounds of interest were dissolved in CH3OH and CH3OD and infused at 20 µL/min into the electrospray source using a Harvard Apparatus 22 syringe pump. ESI mass spectra were obtained on a Finnigan TSQ-700 triple-quadrupole mass spectrometer equipped with an electrospray source. The mass spectra were acquired at 4.2 s/scan, and 4-7 scans were averaged. Capillary temperatures of 125, 150, 175, 200, 225, and 250 °C were used. Spray voltage was 6.0 kV. Dry nitrogen was used as the sheath gas and auxiliary gas for the analysis. During acquisition of the product ion scans, collision energy was 29 eV using argon gas. Important electrospray conditions with regard to in-source dissociation include capillary temperature, tube lens voltage, capillary voltage, and octapole offset voltage. The tube lens voltage, capillary voltage, and octapole offset voltage varied with m/z over (30) McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra; University Science Books: Mill Valley, CA, 1993; p 55.
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Figure 1. Electrospray mass spectrum of BCHA at 10 µg/mL in CH3OH.
the course of a scan but did not exceed 62, 16, and 3 V, respectively, over the mass range of interest. Compounds were purchased from Aldrich Chemical Co. RESULTS To demonstrate the sensitivity and validity of this application, two commercially available compoundssN-tert-butylcyclohexylamine (BCHA) and cyclohexylamine (CHA)swere chosen as examples. The ions at m/z 156 and 100 correspond to protonated BCHA and protonated CHA, respectively. Shown in Figure 1 is the electrospray MS of BCHA at 10 µg/mL in methanol acquired at a capillary temperature of 150 °C. The relative abundance of m/z 100 at a capillary temperature of 150 °C is 3.5%. The lowerthan-normal capillary temperature was used to collect the data in order to limit fragmentation. The CID of m/z 156 produces ions that correspond to the following transitions:
Note that m/z 100 is protonated CHA. This is the same ion structure generated when CHA is electrosprayed from solution. Protonation is located at the primary amine, since this is the functional group with the highest proton affinity and there are no other functional groups in CHA where a proton would readily bind. The CID spectrum of m/z 100, species 1, obtained by tandem
mass spectrometry produces the same fragment ions (m/z 83 and 55) with the same relative abundance whether this ion originates
Figure 2. Electrospray mass spectrum of BCHA at 10 µg/mL in CH3OD.
Figure 4. Electrospray mass spectrum of BCHA at 10 µg/mL and CHA at 1 µg/mL in CH3OD.
Figure 3. CID mass spectrum of m/z 158, the base peak of BCHA in CH3OD.
from electrospraying a solution of CHA or from in-source dissociation of protonated BCHA. Therefore, it is unknown whether the ion at m/z 100 shown in Figure 1 is due to a fragment ion generated from in-source dissociation of m/z 156 (protonated BCHA) or a low-level impurity of CHA in the sample. Shown in Figure 2 is the electrospray mass spectrum of BCHA at 10 µg/mL in CH3OD. The base peak, (MD + D)+, is shifted up 2 Da to m/z 158 due to the exchange of two hydrogen atoms for deuterium atoms. The ion at m/z 100 is also shifted up 2 Da to m/z 102. The proposed structure of m/z 102 is shown in Figure 2 (inset). Shown in Figure 3 is the CID spectrum of m/z 158, along with the proposed fragment ion structures. The ion at m/z 102 is consistent with the loss of 2-methylpropene from m/z 158. In this case, the leaving group transfers a (nonactive) hydrogen to the fragment ion. The electrospray MS of BCHA at 10 µg/mL and CHA at 1 µg/mL in CH3OD is shown in Figure 4. With the addition of CHA, there is an increase in the abundance of the peak at m/z 103, species 2, which would correspond to (MD + D)+ of CHA. This proves that the ion at m/z 102, species 3, is due to a fragment ion and not to a CHA impurity.
The electrospray MS of BCHA at 10 µg/mL and CHA at 0.1 µg/mL in CH3OD is shown in Figure 5. In this case, the ratio of
Figure 5. Electrospray mass spectrum of BCHA at 10 µg/mL and CHA at 0.1 µg/mL in CH3OD.
m/z 103 to 102 is about 18%, which is above the ratio of the natural isotopic abundance of 3. This is proof that an impurity as low as 1% (w/w) can be discerned from a fragment ion with the same structure. Shown in Figure 6A is the CID mass spectrum of m/z 102, species 3 (see Figure 5). It loses NHD2 to give m/z 83, which is a monoisotopic ion (12C6H11+) and should not (and does not) have an isotopic distribution. Shown in Figure 6B is the CID mass spectrum of m/z 103 (Figure 5), which contains two species: deuterated CHA (2) and fragment ion 3, which contains one 13C atom. 2 loses ND3 to give m/z 83 (only), and 13C-labeled 3 loses NHD2 to give m/z 84 only. The ion at m/z 83 is a first-generation product ion of 2, deuterated CHA. The ion at m/z 84 is a second-generation product ion, cyclohexane cation containing one 13C atom (13C12C5H11). The product ion spectra shown in Figure 6 are further proof that a 1% impurity with the same chemical structure as a fragment ion can be differentiated using this method. The preceding examples were used to clearly delineate the process of using H/D exchange to differentiate fragment ions from pseudomolecular ions with the same structure. The analysis was then performed on a compound synthesized in our laboratory. Shown in Figure 7 is the electrospray mass spectrum of the fluoronaphthylamine in methanol acquired at a capillary temperature of 125 °C. The base peak is due to the protonated molecule. The next largest ion at m/z 236 could be a fragment ion or the corresponding protonated primary amine. The CID spectrum of Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
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Figure 8. Electrospray mass spectrum of fluoronaphthylamine in CH3OD.
Figure 6. CID mass spectrum of (A) m/z 102 and (B) m/z 103 from Figure 5.
Figure 7. Electrospray mass spectrum of fluoronaphthylamine in CH3OH.
m/z 292 produces m/z 236, 201, and 74. The electrospray MS obtained when this compound was dissolved in CH3OD is shown in Figure 8. The base peak is shifted up 3 Da to m/z 295, which is in agreement with the two active hydrogens present in the molecule. The ion 4 at m/z 236 is shifted up to m/z 239. This indicates that it is a fragment ion, because the completely exchanged primary amine will produce ion 5 at m/z 240.
It should be noted that, during the acquisition of the MS data, the parameters that influence dissociation in the source were maintained at values that would limit the amount of energy 1528 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
received by the analyte ion; the source conditions were not optimized for best sensitivity or greatest signal. This was done to limit the amount of fragmentation in the source during desolvation and illustrate that fragmentation in the source can occur even when using gentle electrospray ionization conditions. When higher capillary temperatures, tube lens voltage, capillary voltage, and octapole offset voltage were used, a greater ion current was detected for the compound of interest. However, the relative abundance of fragment ions due to in-source dissociation was greater when source conditions were optimized for best sensitivity. The finalized electrospray conditions utilized represent a balance between the desire to eliminate in-source dissociation and provide adequate sensitivity. DISCUSSION Successful fragment ion identification for species 3 and 4 relies on the contingency that there is no exchange of active hydrogens on the fragment ions with evaporated solvent molecules (CH3OD). Under the conditions described here, no experimental evidence of gas-phase H/D exchange with fragment ions was detected. The three primary reasons H/D exchange was not observed for fragment ions 3 and 4 (in the gas phase with CH3OD) are believed to be the following: First, Hunt and Sethi have shown that the proton affinity difference between the analyte and the reagent gas is a major factor in determining the degree of exchange in chemical ionization mass spectrometry.31 Ion cyclotron resonance (ICR) investigations by researchers have shown that, for protonated compounds, H/D exchange reactions are limited when the proton affinity of the neutral base is greater than that of the deuterated reagent by more than 20 kcal/mol.32,33 Isotopic exchange of protonated aromatic compounds with deuterated gases (3 mTorr) in the collision cell of a tandem mass spectrometer has been studied. Under these conditions, it was determined that CH3OD exchanged specific types of active hydrogens, such as the acidic hydrogens of phenols and carboxylic acids. However, the amino hydrogens did not exchange with CH3OD, even with trapping of mass-selected ions in the collision region of a triple-quadrupole mass spectrometer.34 These findings are in agreement with those (31) Hunt, D. F.; Sethi, S. K. J. Am. Chem. Soc. 1980, 102, 6953-6963. (32) Ausloos, P.; Lias, S. G. J. Am. Chem. Soc. 1977, 99, 4198-4199. (33) Ausloos, P.; Lias, S. G. J. Am. Chem. Soc. 1981, 103, 3641-3647. (34) Ranasinghe, A.; Cooks, R. G.; Sethi, S. K. Org. Mass Spectrom. 1992, 27, 77-88.
presented here with regard to the absence of H/D exchange of fragment ions in the gas phase. Thus, the proton affinity difference between CH3OD and the primary amine moiety in species 3 and 4 is a major factor preserving the lone unexchanged active hydrogen in the fragment ions. Limited H/D exchange reactions have been reported when the gas-phase basicity of the compound is significantly larger, 20 kcal/mol. Using a four-sector instrument equipped with a highpressure collision cell, Cheng and Fenselau observed such gasphase H/D exchange of protonated peptide ions.35 Under highpressure conditions, they found that H/D exchange reactions occurred even with functional groups having proton affinities in excess of 20 kcal/mol. Gard et al. have also reported gas-phase H/D exchange kinetics of several protonated amino acids and dipeptides in an external source Fourier transform mass spectrometer (FTMS).36,37 Gas-phase H/D exchange for all functional groups was not complete using trapping times up to 15 s. However, the ion environment of the FTMS and the use of a highpressure collision cell are not representative of the conditions used to acquire these data. Second, the amount of dry nitrogen (about 5 L/min is the sheath and auxiliary gas flow rate) entering the source canister is in about a 400:1 molar ratio compared to the amount of CH3OD (20 µl/min). Thus, the collision gas where fragmentation occurs (between the exit of the heated capillary and the octapole) is made up primarily of N2. Therefore, most collisions encountered by fragment ions should be with N2 (and not CH3OD). Collisions with N2 would not lead to H/D exchange. Third, the fragmentation of the analyte ion occurs after desolvation in a medium- to low-pressure area of the source. The approximate pressure in the region just between the heated capillary and the skimmer is about 0.5-1 Torr, and the pressure between the skimmer and the octapole is about 1 mTorr. The physical dimensions of these regions are small (shorter than most collision cells). The short distance, small time scale, and relatively low pressures in these regions limit the number of effective collisions in which H/D exchange of a fragment ion can transpire. Both CH3OH and CH3OD noncovalent adducts were detected in the electrospray spectra of fluoronaphthylamine. The gentle electrospray conditions preserved the methanol noncovalent binding to fluoronaphthylamine, yet there is some in-source dissociation of the protonated molecule. As previously mentioned, the fragment ions that can be differentiated by H/D exchange are usually even-electron ions, which are the equivalent of a protonated molecule. A fragment ion detected in the CID mass spectrum of m/z 158, (MD + D)+ of BCHA, is m/z 83. In theory, H/D exchange could be used to determine if m/z 83 is a fragment ion or a protonated molecule. An ion with a relative abundance of less than 1% was detected in the electrospray mass spectrum of BCHA when dissolved in CH3OH or CH3OD (see Figures 1 and 2). When cyclohexene is protonated from CH3OH, the following transition occurs, and an ion at m/z 83 is produced: (35) Cheng, X.; Fenselau, C. Int. J. Mass Spectrom. Ion Processes 1992, 122, 109-119. (36) Gard, E.; Willard, D.; Bregar, J.; Green, M. K.; Lebrilla, C. B. Org. Mass Spectrom. 1993, 28, 1632-1639. (37) Gard, E.; Green, M. K.; Bregar, J.; Lebrilla, C. B. J. Am. Soc. Mass Spectrom. 1994, 5, 623-631.
However, cyclohexene deuterated from CH3OD would produce an ion at m/z 84 based on the following transition:
Note that the fragment ion, m/z 83, does not shift in mass whether BCHA is electrosprayed from CH3OH or CH3OD; a deuterated cyclohexane cation would shift up 1 Da to m/z 84. Thus, a deuterated cyclohexane cation could be differentiated from the fragment ion by H/D exchange. However, the electrospray response for cyclohexene is poor, and it is improbable that it could be detected. Therefore, the degree of confidence that m/z 83 is a fragment ion and not protonated cyclohexene is high without the use of H/D exchange. The analogous reasoning could be used to exclude the ion at m/z 57 (in the CID spectrum of BCHA, see Figure 3) as protonated 2-methylpropene. With regard to this application and the fragmentation of peptides, the y ions are important for determining the C-terminal sequence. In the CID formation of y ions, the amide bond is cleaved, and two hydrogen atoms are transferred to the C-terminal fragment. It has been demonstrated using deuterium labeling that an active hydrogen migrates during cleavage of the amide bond.7,13 This transfer of an active hydrogen in the formation of the y ion means that all active hydrogens are exchanged. This renders impossible differentiation between the y fragment ions and molecular ions. CONCLUSION For many compounds, low-abundance fragment ions generated from in-source dissociation are detectable even using gentle electrospray conditions. Since most CID involves the loss of evenelectron neutral species, the even-electron fragment ion cannot be distinguished from protonated molecules in many cases. Electrospraying the sample from a protic deuterated solvent will differentiate fragment ions from pseudomolecular ions when the active hydrogens of the fragment ion are not fully exchanged for deuterium. An example of this is a fragment ion of BCHA which has the same structure as protonated CHA. H/D exchange allowed the identification of CHA in a sample of BCHA at a level of 1% (w/w) without the use of chromatography. The use of H/D exchange can differentiate fragment ions from pseudomolecular ions in many cases and preclude the necessity of developing chromatography-based methods in such analysis. ACKNOWLEDGMENT The authors thank Bart Emary for his comments in the preparation of this paper. Received for review October 9, 1996. Accepted January 28, 1997.X AC961030G X
Abstract published in Advance ACS Abstracts, March 15, 1997.
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